Tatyana A. Sysoeva

Engagement of Arginine Finger to ATP Triggers Large Conformational Changes in NtrC1 AAA+ ATPase For Remodeling Bacterial RNA Polymerase

The NtrC-like AAA+ ATPases control virulence and other important bacterial activities through delivering mechanical work to σ54-RNA polymerase to activate transcription from σ54-dependent genes. We report the first crystal structure for such an ATPase, NtrC1 of Aquifex aeolicus, in which the catalytic arginine engages the γ-phosphate of ATP. Comparing the new structure with those previously known for apo and ADP-bound states supports a rigid-body displacement model that is consistent with large-scale conformational changes observed by low-resolution methods. First, the arginine finger induces rigid-body roll, extending surface loops above the plane of the ATPase ring to bind σ54. Second, ATP hydrolysis permits Pi release and retraction of the arginine with a reversed roll, remodeling σ54-RNAP. This model provides a fresh perspective on how ATPase subunits interact within the ring-ensemble to promote transcription, directing attention to structural changes on the arginine-finger side of an ATP-bound interface.

Antibiotics as a selective driver for conjugation dynamics

It is generally assumed that antibiotics can promote horizontal gene transfer. However, because of a variety of confounding factors that complicate the interpretation of previous studies, the mechanisms by which antibiotics modulate horizontal gene transfer remain poorly understood. In particular, it is unclear whether antibiotics directly regulate the efficiency of horizontal gene transfer, serve as a selection force to modulate population dynamics after such gene transfer has occurred, or both. Here, we address this question by quantifying conjugation dynamics in the presence and absence of antibiotic-mediated selection. Surprisingly, we find that sublethal concentrations of antibiotics from the most widely used classes do not significantly increase the conjugation efficiency. Instead, our modelling and experimental results demonstrate that conjugation dynamics are dictated by antibiotic-mediated selection, which can both promote and suppress conjugation dynamics. Our findings suggest that the contribution of antibiotics to the promotion of horizontal gene transfer may have been overestimated. These findings have implications for designing effective antibiotic treatment protocols and for assessing the risks of antibiotic use.

Nucleotide-induced asymmetry within ATPase activator ring drives σ54–RNAP interaction and ATP hydrolysis

It is largely unknown how the typical homomeric ring geometry of ATPases associated with various cellular activities enables them to perform mechanical work. Small-angle solution X-ray scattering, crystallography, and electron microscopy (EM) reconstructions revealed that partial ATP occupancy caused the heptameric closed ring of the bacterial enhancer-binding protein (bEBP) NtrC1 to rearrange into a hexameric split ring of striking asymmetry. The highly conserved and functionally crucial GAFTGA loops responsible for interacting with σ54-RNA polymerase formed a spiral staircase. We propose that splitting of the ensemble directs ATP hydrolysis within the oligomer, and the rings asymmetry guides interaction between ATPase and the complex of σ54 and promoter DNA. Similarity between the structure of the transcriptional activator NtrC1 and those of distantly related helicases Rho and E1 reveals a general mechanism in homomeric ATPases whereby complex allostery within the ring geometry forms asymmetric functional states that allow these biological motors to exert directional forces on their target macromolecules.

Dimer recognition and secretion by the ESX secretion system in Bacillus subtilis

Significance Bacteria use a wide range of secretion mechanisms to export proteins from the cytoplasm. These secretion pathways differ in the nature of substrate recognition signal and the mode of substrate translocation. This work elucidates fundamental properties of substrate secretion by the early secretory antigen 6 kDa (ESX) system. We use systematic mutagenesis and targeted crosslinking of an ESX substrate from Bacillus subtilis to provide, to our knowledge, the first experimental evidence for secretion of an intact dimeric complex requiring a composite recognition signal formed by both members of the complex. Protein secretion typically involves translocation of unfolded polypeptides or transport of monomeric folded proteins. Here we provide, to our knowledge, the first experimental evidence for secretion of an intact multimeric complex requiring a signal formed by both members of the complex. Using systematic mutagenesis of a substrate involved in early secretory antigen 6 kDa (ESX) secretion in Bacillus subtilis, we demonstrate that export of the substrate requires two independent motifs. Using mixed dimers, we show that these motifs must form a composite secretion signal in which one motif is contributed by each subunit of the dimer. Finally, through targeted crosslinking we show that the dimer formed in the cell is likely secreted as a single unit. We discuss implications of this substrate recognition mechanism for the biogenesis and quality control of secretion substrates and describe its likely conservation across ESX systems.

Linked Domain Architectures Allow for Specialization of Function in the FtsK/SpoIIIE ATPases of ESX Secretion Systems

Among protein secretion systems, there are specialized ATPases that serve different functions such as substrate recognition, substrate unfolding, and assembly of the secretory machinery. ESX (early secretory antigen target 6 kDa secretion) protein secretion systems require FtsK/SpoIIIE family ATPases but the specific function of these ATPases is poorly understood. The ATPases of ESX secretion systems have a unique domain architecture among proteins of the FtsK/SpoIIIE family. All well-studied FtsK family ATPases to date have one ATPase domain and oligomerize to form a functional molecular machine, most commonly a hexameric ring. In contrast, the ESX ATPases have three ATPase domains, encoded either by a single gene or by two operonic genes. It is currently unknown which of the ATPase domains is catalytically functional and whether each domain plays the same or a different function. Here we focus on the ATPases of two ESX systems, the ESX-1 system of Mycobacterium tuberculosis and the yuk system of Bacillus subtilis. We show that ATP hydrolysis by the ESX ATPase is required for secretion, suggesting that this enzyme at least partly fuels protein translocation. We further show that individual ATPase domains play distinct roles in substrate translocation and complex formation. Comparing the single-chain and split ESX ATPases, we reveal differences in the requirements of these unique secretory ATPases.

Regulation and action of the bacterial enhancer-binding protein AAA+ domains.

Bacterial EBPs (enhancer-binding proteins) play crucial roles in regulating cellular responses to environmental changes, in part by providing efficient control over sigma(54)-dependent gene transcription. The AAA+ (ATPase associated with various cellular activites) domain of the EBPs, when assembled into a ring, uses energy from ATP binding, hydrolysis and product release to remodel the sigma(54)-RNAP (RNA polymerase) holoenzyme so that it can transition from closed to open form at promoter DNA. The assembly, and hence activity, of these ATPases are regulated by many different signal transduction mechanisms. Recent advances in solution scattering techniques, when combined with high-resolution structures and biochemical data, have enabled us to obtain mechanistic insights into the regulation and action of a subset of these sigma(54) activators: those whose assembly into ring form is controlled by two-component signal transduction. We review (i) experimental considerations of applying the SAXS (small-angle X-ray scattering)/WAXS (wide-angle X-ray scattering) technique, (ii) distinct regulation mechanisms of the AAA+ domains of three EBPs by similar two-component signal transduction receiver domains, and (iii) major conformational changes and correlated sigma(54)-binding activity of an isolated EBP AAA+ domain in the ATP hydrolysis cycle.

Structural characterization of the late competence protein ComFB from Bacillus subtilis.

Many bacteria take up DNA from their environment as part of the process of natural transformation. DNA uptake allows microorganisms to gain genetic diversity and can lead to the spread of antibiotic resistance or virulence genes within a microbial population. Development of genetic competence (Com) in Bacillus subtilis is a highly regulated process that culminates in expression of several late competence genes and formation of the DNA uptake apparatus. The late competence operon comF encodes a small protein of unknown function, ComFB. To gain insight into the function of ComFB, we determined its 3D structure via X-ray crystallography. ComFB is a dimer and each subunit consists of four α-helices connected by short loops and one extended β-strand-like stretch. Each subunit contains one zinc-binding site formed by four cysteines, which are unusually spaced in the primary sequence. Using structure- and bioinformatics-guided substitutions we analyzed the inter-subunit interface of the ComFB dimer. Based on these analyses, we conclude that ComFB is an obligate dimer. We also characterized ComFB in vivo and found that this protein is produced in competent cells and is localized to the cytosol. Consistent with previous reports, we showed that deletion of ComFB does not affect DNA uptake function. Combining our results, we conclude that ComFB is unlikely to be a part of the DNA uptake machinery under tested conditions and instead may have a regulatory function.

Dissecting the effects of antibiotics on horizontal gene transfer: Analysis suggests a critical role of selection dynamics.

Horizontal gene transfer (HGT) is a major mechanism responsible for the spread of antibiotic resistance. Conversely, it is often assumed that antibiotics promote HGT. Careful dissection of the literature, however, suggests a lack of conclusive evidence supporting this notion in general. This is largely due to the lack of well-defined quantitative experiments to address this question in an unambiguous manner. In this review, we discuss the extent to which HGT is responsible for the spread of antibiotic resistance and examine what is known about the effect of antibiotics on the HGT dynamics. We focus on conjugation, which is the dominant mode of HGT responsible for spreading antibiotic resistance on the global scale. Our analysis reveals a need to design experiments to quantify HGT in such a way to facilitate rigorous data interpretation. Such measurements are critical for developing novel strategies to combat resistance spread through HGT.

ADPase activity of recombinantly expressed thermotolerant ATPases may be caused by copurification of adenylate kinase of Escherichia coli

Except for apyrases, ATPases generally target only the γ‐phosphate of a nucleotide. Some non‐apyrase ATPases from thermophilic microorganisms are reported to hydrolyze ADP as well as ATP, which has been described as a novel property of the ATPases from extreme thermophiles. Here, we describe an apparent ADP hydrolysis by highly purified preparations of the AAA+ ATPase NtrC1 from an extremely thermophilic bacterium, Aquifex aeolicus. This activity is actually a combination of the activities of the ATPase and contaminating adenylate kinase (AK) from Escherichia coli, which is present at 1/10 000 of the level of the ATPase. AK catalyzes conversion of two molecules of ADP into AMP and ATP, the latter being a substrate for the ATPase. We raise concern that the observed thermotolerance of E. coli AK and its copurification with thermostable proteins by commonly used methods may confound studies of enzymes that specifically catalyze hydrolysis of nucleoside diphosphates or triphosphates. For example, contamination with E. coli AK may be responsible for reported ADPase activities of the ATPase chaperonins from Pyrococcus furiosus, Pyrococcus horikoshii, Methanococcus jannaschii and Thermoplasma acidophilum; the ATP/ADP‐dependent DNA ligases from Aeropyrum pernix K1 and Staphylothermus marinus; or the reported ATP‐dependent activities of ADP‐dependent phosphofructokinase of P. furiosus. Purification methods developed to separate NtrC1 ATPase from AK also revealed two distinct forms of the ATPase. One is tightly bound to ADP or GDP and able to bind to Q but not S ion exchange matrixes. The other is nucleotide‐free and binds to both Q and S ion exchange matrixes.

Universal antibiotic tolerance arising from antibiotic-triggered accumulation of redox metabolites

Pseudomonas aeruginosa is an opportunistic pathogen that often infects open wounds or patients with cystic fibrosis. Once established, P. aeruginosa infections are notoriously difficult to eradicate. This difficulty is in part due to the ability of P. aeruginosa to tolerate antibiotic treatment at the individual-cell level or through collective behaviors. Here we describe a new mechanism by which P. aeruginosa tolerates antibiotic treatment by modulating its global cellular metabolism. In particular, treatment of P. aeruginosa with sublethal concentrations of antibiotics covering all major classes promoted accumulation of the redox-sensitive phenazine - pyocyanin (PYO). PYO in turn conferred general tolerance against diverse antibiotics for both P. aeruginosa and other Gram-negative and Gram-positive bacteria. We show that PYO promotes energy generation to enhance the activity of efflux pumps, leading to enhanced antibiotic tolerance. This property is shared by other redox-active phenazines produced by P. aeruginosa. Our discovery sheds new insights into the physiological functions of phenazines and has implications for designing effective antibiotic treatment protocols. Author Summary Antibiotic tolerance can facilitate the evolution of resistance, and here we describe a previously unknown mechanism of collective antibiotic tolerance in Pseudomonas aeruginosa. In particular, P. aeruginosa treated with sublethal concentrations of antibiotics covering all major classes promotes accumulation of pyocyanin (PYO), an important virulence factor. In turn, PYO confers general tolerance against diverse antibiotics for both P. aeruginosa and other bacteria. Our discovery is a perfect example of what Nietzsche once said: That which does not kill me makes me stronger.